1. Field of the Invention
The present invention relates generally to the field of biology. More particularly, it concerns methods for inhibiting fibrin clot formation and the use of decorin, or related peptides as anticoagulating agents.
SLRPs encompass a class of secreted proteoglycans that include five structurally related but genetically distinct members: decorin, biglycan, fibomodulin, lumincan, and epiphycan, which was originally called PG-Lb. These proteoglycans share the unique feature of being composed primarily of leucine-rich tandem repeats that confer most of the biological functions. A close examination of the overall protein core structure reveals that it consists of three main regions: an amino-terminal region, which contains the negatively charged GAGs or tyrosine sulfate; a central domain with varying numbers of LRRs; and a carboxyl end region of poorly defined function. In all cases, the central domain is flanked by cysteine-rich clusters. In decorin, biglycan, and epiphycan, the amino-terminal region harbors 1-2 GAG chains that can be either dermatan or chondroitin sulfate.
2. Description of Related Art
Decorin is composed of a 40 kD core protein and usually carries a chondroitin sulfate/dermatan sulfate glycosaminoglycan (GAG) chain. Many GAGs effect blood coagulation activity. For example, heparin, and heparan sulfate (HS) have been used as efficient anticoagulants clinically for decades. Dermatan sulfate (DS) is also a potent anticoagulant both in vivo and in vitro (Brister, 1994; Linhardt, 1994; Lacoviello, 1996). However, the anticoagulation mechanisms of heparin/HS and DS are different. Heparin and HS both accelerate the activity of antithrombin III as it inhibits the activity of thrombin. Dermatan sulfate GAG, on the other hand, accelerates the inhibition activity of heparin cofactor II to thrombin (Tollefsen, 1983; Maimone, 1990). Antithrombin III and heparin cofactor II are inhibitors of serine proteases that inhibit blood coagulation by inhibiting the activity of thrombin and related serine proteases. Dermatan sulfate GAG isolated from decorin has anticoagulant activity, enhancing the inhibitory activity of heparin cofactor II to thrombin (Whinna, 1993). In contrast, both hyaluronic acid (HA) and chondroitin (CS) bind to the plasma protein fibrinogen and induce fibrin polymerization, resulting in decreased clotting time (LeBoeuf, 1987). The traditional anticoagulant heparin and the recent dermatan sulfate GAGs have been used in treating the patients and animals with thrombosis (Brister, 1994; Linhardt, 1994; Lacoviello, 1996). However, the side effect of hemorrhage has been a problem (Matthiasson, 1995).
Decorin is distributed in a variety of tissues such as skin, bone, cartilage, tendon, cornea, and blood vessel (lozzo, 1996). Decorin interacts with a variety of extracellular matrix (ECM) molecules such as collagens, fibronectin, and transforming growth factor xcex2 and regulates their biological functions in matrix assembly, cell adhesions, and signaling for cell proliferation and differentiation. These interactions are believed to be important in embryonic development, wound healing processes, as well as in pathogenic conditions such as atherosclerosis and tumorgenesis.
Decorin gene knock out mice have an abnormal arrangement of collagen fibrils which results in fragile skin (Danielson, 1997). In addition, decorin binds to several collagens, for example, type I, II, III, V, VI, and XIV through core protein or GAG chains, as shown in vitro binding studies (Pogany, 1992; Hedbom, 1993; Ramamurthy, 1996; Bidanset, 1992; Font, 1996; Font, 1993). Decorin associates with collagen fibers and regulates collagen fibrillogenesis (Rada, 1993; Brown, 1989, Vogel, 1984). The binding of decorin to collagen type I is mediated by the decorin core protein (Schxc3x6nherr, 1995; Svensson, 1995; Dresse, 1997). Molecular modeling predicts that the ten LRRs of decorin core protein form a horse shoe like structure that accommodates a single type I collagen triple helix (Weber, 1996). Both in vitro and in vivo studies suggest that decorin is important in regulating the arrangement of collagen fibrils involved in matrix assembly.
Decorin interacts with other ECM molecules. For example, decorin binds to fibronectin with high affinity via its core protein (Schmidt, 1987; Schmidt, 1991). The interaction of decorin to fibronectin is affected in cell adhesion (Lewandowska, 1987; Bidanset, 1992). Soluble molecules in the ECM, such as a variety of growth factors, ions, and water, are important in cell signaling, controlling cell growth, and programming cell death. Decorin binds transforming growth factor xcex2 (TGFxcex2) and regulates its function in cell proliferation and differentiation (Hildebrand, 1994; Yamaguchi, 1990). Expression of decorin in colon carcinoma cells may inhibit cell growth by regulating the epidermal growth factor (EGF) signaling pathway (Moscatello, 1998; Patel, 1998). Additionally, decorin may bind to human complement C1q and inhibit activity of the C1 complex involved in inflammatory cascade (Krumdieck, 1992).
Zinc, a divalent cation (Zn2+), plays important biological roles in the catalytic activity of enzymes and in the stabilization of protein structures. For example, metalloproteases require zinc ions to be active. Also, the zinc finger containing transcription factor complexes Zn2+ to form a structure suitable to bind with DNA. The N-terminal cysteine containing domain of decorin binds Zn2+ ions with high affinity, KD=3xc3x9710xe2x88x927 M, suggesting that Zn2+ will interact with decorin in vivo (Yang, 1999). In addition, circular dichroism experiments indicate that Zn2+ ions alter the secondary structure of the recombinant decorin N-terminal peptide (Yang, 1999). However, the function of decorin-Zn2+ complex is unclear.